Despite the fact that intracellular pathogens that are confined within host cell phagosomes are able to induce CD8+ T cells in vitro and in vivo, and in the case of Leishmania this response is required for protection, the mechanisms controlling the cross-presentation of phagosome-derived antigens are poorly understood.To facilitate the study of cross-presentation of Leishmania antigens, we engineered L. major parasites to express a fragment of ovalbumin containing the SIINFEKL epitope recognized by TCR transgenic OT-I CD8+ T cells, as part of a parasite 3? nucleotidase protein. In vivo and in vitro, both secreted and cytosolic forms of NT-OVA were efficiently cross-presented to CD8+ OT-I T cells. The presentation of the SIINFEKL peptide by L. major NT-OVA-infected DC in vitro or by APCs involved in cross-priming in vivo, was not affected by the absence of TAP1, suggesting that the conventional MHC class I processing pathway is not used for this particular epitope. When we extended these studies to the cross-presentation of wild-type L. major antigens recognized by a polyclonal population of in vivo primed CD8+ T cells, a dispensable role for TAP1 was still observed. Furthermore, TAP1 deficient mice infected with a low dose of L. major in the ear were able to control infection as efficiently as wild type mice. In parallel studies, we observed that cross presentation of OVA secreted by transfected Toxoplasma gondii was strictly TAP dependent in vitro and in vivo. Thus in the case of Leishmania, exogenous antigen processing and presentation does not appear to follow a phagosome-to-cytosol pathway as it has been reported for latex beads and other intracellular microorganisms. A role for lysosomal hydrolases in the processing of Leishmania antigens was indicated by the strong inhibition of cross-priming observed using leupeptin treated DC. The L. major murine model has been useful to emphasize the importance of Th1/Th2 balance in resistance or susceptibility to Leishmania infection. However, Th2 polarization, as occurs during L. major infection in BALB/c mice, has failed to account for severe or non-cure forms of leishmaniasis in humans. The role of other disease promoting factors might be more easily studied in mouse strains that do not maintain a confounding, strong Th2 bias during infection. We have conducted a series of experiments in C57BL/6 mice involving an L. major strain, Seidman (Sd), that in contrast to the majority of L. major isolates, produces non-healing lesions in these mice. In B6 mice that developed non-healing dermal lesions, a polarized IFN-g response, with no detectable IL-4, was measured in the CD4+ T cells from the lesion and draining node, and the magnitude of this response even exceeded that seen in the mice infected with a healing strain of L. major. Thus Th1 response defects and Th2 immune deviation cannot explain the failure of these mice to control infection. A role for CD4+CD25+ regulatory T cells was indicated by the results of CD25 depletion experiments, which increased parasite killing in the site. The treatment of mice with IL-10R antibody had an even more pronounced effect than CD25+ depletion on promoting healing and parasite clearance. Thus Treg and IL-10 are essential susceptibility factors in this model, and their effects seem not so much to inhibit Th1 priming as to suppress the expression of effector activities in the inflammatory site. We observed in our studies of Sd infection in B6 mice that despite their inability to heal their primary site of infection, when the animals were re-challenged in the contra-lateral ear, they were completely protected. Thus the Treg and other suppressive mechanisms that function within the primary inflammatory site to prevent effective clearance by Th1 cells do not inhibit the expression of Th1 effector activity in a naive rechallenge site. We have argued that the activation of Treg during infection provides a benefit to the host by not only controlling the severity of inflammation, but by maintaining a chronic infection the Treg will also maintain an effector memory pool necessary for resistance to reinfection. Since individuals with healed lesions and presumed persistent infection have life long immunity to reinfection, vaccination using virulent L. major promastigotes, termed leishmanization, remains the gold standard in terms of the potency and durability of acquired immunity that can be achieved, but remains problematic because of the severity of the vaccination-lesions. In an attempt to develop leishmanization protocols that minimize pathology while maintaining long term protection, live parasites have been co-injected with TLR agonists, either CpG ODNs or imiguimod, into Rhesus monkeys. The CpG ODN (D class) provoked a transient inflammation but reduced the size of the cutaneous lesion that developed. Thus, immune-modulation using CpG ODNs might be a practical approach to improving the safety of a highly effective live vaccine that has already been widely applied.